6470 triple quadrupole tandem mass spectrometer

For quantification of thyroid hormone levels, tissues were dissected, snap frozen in liquid nitrogen, and stored at-80 °C. For brain analysis, after homogenization of whole-brains at around −200 °C, about 100 mg was used for LC-MS/MS quantification of thyroid hormones (TH) using the Agilent 1290 Infinity II LC system interfaced with an Agilent 6470 triple quadrupole tandem mass spectrometer as described previously [20 ]. For the analysis of liver, heart, BAT and eWAT a nanoAcquity system interfaced with a QTof2 mass detector was used as previously described [21 ,22 (link)].

LC–MS/MS (Agilent) was performed as previously described (8 (link)) using an Agilent 1200 series LC and 6470 triple quadrupole tandem mass spectrometer. In brief, DMP was quantified using m/z 97.4→56.2 and qualified using m/z 97.4→70.2, and 3,4-HDMP used m/z 99.2→57.2. The same Phenomenex Kinetex biphenyl column (50 × 3 mm, 2.6 µm) was used, and a second Phenomenex Kinetex C18 column (50 × 3 mm, 2.6 µm) was also used to identify whether the stationary phase played a specific role in the chromatography. Perfluorooctanoic acid (0.01%) in water (phase A) and methanol (phase B) was used for quantification and the flow rate was optimized to 0.4 mL/min, with an injection volume of 1 µL. The gradient started with 20% phase B for 0.5 min and then increased to 80% by 4 min, held until 7.5 min and then returned to 20%.